Encoding Long Term Stability of Neuronal Function

描述（由申请人提供）：在胚胎发育期间，转录因子对于建立决定神经元前体和神经元命运的密码至关重要。然而，大多数负责神经元功能特性的蛋白质的半衰期从几分钟到几个小时不等;因此，它们必须在神经发育结束后很长时间内受到严格调控，以维持神经元功能。令人惊讶的是，很少有人知道转录因子在这个过程中发挥的作用。我们假设，转录因子，最初被表征为早期神经发育基因，需要在完全发育的运动神经元（MN），以维持神经元的功能。 我们建议彻底影响水平的三个转录因子gsb，isl和eve内完全发达的MN和确定它们控制的神经元功能。本研究的长期目标是建立这些转录因子的分子靶点。果蝇的神经肌肉接头（NMJ）提供了一个模型，非常适合于充分发展的MN的研究。MN已经到达它们的肌肉目标，并且在胚胎发生结束之前释放神经递质。我们可以在它们完全发育后4到5天，即接近幼虫生命结束时，测试MN的活力，突触结构和生理学，比平均蛋白质的寿命长许多倍。此外，转基因RNAi和条件表达的使用允许在完全发育的MN中敲除基因表达，从而绕过胚胎对转录因子的需求。我们最近表明，gsb是普遍表达在发育完全的MN的晚期幼虫中枢神经系统。使用免疫组织化学，我们将首先表明，eve和isl在一个子集的充分发展的MN内表达。在第二个目标中，我们将结合联合收割机经典遗传学和Gal 4/UAS系统与Gal 80 TS的结合使用，以在发育后期敲低或过表达转录因子。然后，我们将使用免疫组织化学和电子显微镜来确定MN的活力或NMJ的结构是否受到完全发育的MN内敲低或过表达eve、isl或gsb的影响。然后，我们将在NMJ进行细胞内电生理记录，以确定是否eve，isl或gsb控制突触释放和稳态可塑性在完全发育的MN。这项研究将提供一个概念性的模板，将功能归因于发育完全的神经元内的转录因子;这反过来又可能导致对神经元衰老和神经元变性的分子过程的深入了解。

DESCRIPTION (provided by applicant): During embryonic development, transcription factors are essential for establishing a code that will determine the fate of neuronal precursors and neurons. However, most proteins that are responsible for a neuron's functional properties have a half-life ranging from minutes to several hours; they therefore must be tightly regulated long after neural development is over in order to maintain neuronal function. Surprisingly, very little is known about the role that transcription factors play in this process. We hypothesize that transcription factors that were initially characterized as early neuro-developmental genes, are required in fully developed motorneurons (MNs) to maintain neuronal function. We propose to drastically affect the levels of three transcription factors gsb, isl and eve within fully developed MNs and determine the neuronal functions they control. The long term goal of this research is to establish the molecular targets of these transcription factors. The Drosophila neuromuscular junction (NMJ) provides a model that is well-suited to the study of fully developed MNs. MNs have reached their muscle targets and are releasing neurotransmitter before the end of embryogenesis. We can test MN viability, synaptic structure and physiology 4 to 5 days after they are fully developed, near the end of the larval life, many times longer than the lifetime of the average protein. Moreover, the use of transgenic RNAi and conditional expression allows for knockout of gene expression in fully developed MNs, therefore bypassing the embryonic requirement for transcription factors. We have recently shown that gsb is ubiquitously expressed in fully developed MNs of the late larval CNS. Using immunohistochemistry, we will first show that eve and isl are expressed within a subset of fully developed MNs. In a second aim we will combine classical genetics and the use of the Gal4/UAS system in conjunction with Gal80TS to knock down or over-express transcription factors late, after development. We will then use immunohistochemistry and electron microscopy to determine whether the viability of the MN or the structure of the NMJ is affected by knocking down or over-expressing eve, isl or gsb within fully developed MNs. We will then perform intracellular electrophysiological recordings at the NMJ to determine whether eve, isl or gsb control synaptic release and homeostatic plasticity within fully developed MNs. This study will provide a conceptual template attributing a function to transcription factors within fully developed neurons; this in turn could lead to great insights into the molecular processes of neuronal aging and neuronal degeneration.